Device for plasma modification--composition and remodeling

ABSTRACT

Compositions and devices are provided for the specific removal of components of plasma in efficient and economical ways. The devices provide for a tortuous path of the plasma through a high surface material to which is bound a binding compound for removal of the fluid component. The devices find particular application with plasma, in diagnosis, therapy, and for production of specific physiologically active materials.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.191,039 filed May 6, 1988, now U.S. Pat. No. 4,963,265.

INTRODUCTION

1. Technical Field

The subject invention is concerned with plasma processing devicesinvolving separation of soluble plasma components and remodeling ofsoluble plasma components.

2. Background

Blood and lymph are the fluid highways of the body. These fluids providefor the transport of nutrients, metabolites, growth factors, hormones,and the like, from one site in the body to another, allowing forproduction of various compounds by cells and tissues in one part of thebody, for the regulation of cells and tissues in another part of thebody. In addition, these fluids allow for removal of waste materials, soas to prevent the accumulation of compounds which could interfere withthe ability of cells and organs to function. Of equal importance is thefact that these fluids also allow for transport of cells of thehematopoietic system throughout the body to fulfill their variegatedfunction, while also bringing a wide variety of materials to cells ofthe hematopoietic system for processing or for inducing a cellularresponse, as in the case of antigens, pathogens, or the like.

In many situations, the interaction between substances in the blood andhematopoietic cells can result in products which may provide fruitfulinformation about the diseased state of the individual, providecompositions of interest in relation to the host or other individuals,or cause adverse affects to the host. There is, therefore, a substantialinterest in accessing these fluids and isolating, identifying orremodeling various components in the blood stream.

Blood, however, is an extraordinarily complex mixture, which may respondto an alien environment in a wide variety of ways. Commonly, blood clotsresult in the stoppage of flow. Where plasma is used, contact withforeign materials can activate various blood components resulting insubstantial changes in the blood composition. While this may not be aproblem in many instances where the blood or plasma is not being reused,when the blood is to be returned to the host, such changes manydetrimentally affect the host and therefore preclude the reuse of theblood.

In many instances, it is desirable to restore a person's blood, such asin plasmapheresis, because of the uncertainties concerning the safety ofthe blood supply, due to hepatitis, HIV, HTLV-I, or the like, or theavailability of the correct blood type.

It is therefore of interest to develop procedures and equipment whichallow for selective treatment of blood or plasma, by modification of thenature and/or amount of components of the blood and saving the blood forrestoration to the host from which the blood was withdrawn. Necessaryfor this purpose is identification of materials, components, andconditions which allow for such selective treatment without significantadverse effects on the blood.

3. Relevant Literature

Descriptions of blood component removal systems may be found in U.S.Pat. Nos. 4,086,924; 4,103,685; 4,223,672; 4,362,155; 4,428,744;4,464,165; 4,540,401; 4,614,513; 4,627,915 and Re 31,688 and EPA 0 082345. References associated with complement activation include Breillattand Dorson, ASAIO J. (1984) 7:57-63 and McLeod et al., Artif. Organs(1983) 7:443-449.

SUMMARY OF THE INVENTION

Devices are provided for the modification of blood or plasma involvingremoval or remodeling of blood components. The devices provide for anextended fluid path through a biocompatible high surface area packing towhich is bound one or more specific binding components for interactingwith one or more components of blood. The device allows for the smoothcontinuous flow of the fluid stream with substantially uniform contactbetween the fluid stream and the substrate-bound binding components. Thedevice also allows for recovery of components of the blood which bind tothe substrate-bound binding components. Particularly, a number ofcomponents produced by molecular biology techniques have been shown tobe useful in blood treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view of a device according to this invention;

FIG. 2 is an exploded perspective view of a box device and its contentsaccording to the invention;

FIG. 3 is a cross-sectional elevation of a tubular device according tothis invention; and

FIG. 4 is a perspective view of the parts of the device of FIG. 3.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and apparatus are provided for treating blood samples involvingreceptor-ligand complex formation on a solid surface which isbiocompatible with blood or blood fluid derivatives. The fluid stream isdirected through an extended, conveniently tortuous, path comprising ahigh surface area substrate to which a member of the specific bindingpair is nondiffusibly bound. By interaction between components of thefluid stream and the bound specific binding component, one or morecomponents of the blood stream will be removed or remodeled, byincreasing or decreasing concentrations of such components or changingratios of such components.

The blood stream may have been pretreated prior to use. The blood mayhave been subject to prior treatment, such as removal of red cells,platelets, white blood cells, or the like, where the resulting fluid maycontain one or more families of cells or be substantially cell-free.Various compounds may be added such as acid-citrate, dextrin or heparin,and the blood stream may be diluted, concentrated, divided into two ormore streams, or augmented with blood or blood component from the sameor different host. The blood to be combined will be syngeneic orallogeneic.

The subject method may be used for a variety of purposes. In particular,members of a specific binding pair homologous to a component of interestmay be employed to reduce the level of the component in the bloodderived fluid. For example, immune complexes may be removed or remodeledby employing protein A, binding fragments thereof, or proteins havinganalogous binding properties, such as antibodies [other bacterial ormammalian F_(C) receptors] and the like. The antibodies may be specificfor an epitope of a constant region isotype, e.g. IgM, IgG, IgA, IgD, orIgE or an epitope that crosses isotypes. Alternatively, antibodiespresent in the blood stream specific for a particular antigen may beremoved by binding the antigen to the surface. Illustrative antigensinclude DNA or other host substances, particularly various factorsinvolved with host responses to a diseased or aberrant state, such astumor necrosis factor (TNF) associated with septic shock, or antibodiesto the acetylcholine receptor in myasthenia gravis. In addition, theremay be an interest in lowering a concentration of a particular componentof the blood stream, such as insulin, neoplastic cells, steroids, e.g.estrogens, cytokines, lymphokines and other chemotherapeutic agents orbiologicals employed as therapeutic agents. Thus, by varying the bindingcomponent present on the surface, the nature of the fluid stream may bemodified in a single or multiple ways.

The fluid stream is directed, conveniently by pumping, through anextended path, conveniently a tortuous path, so as to expose the streamto a high level of binding component, while substantially minimizingadverse effects on the properties of the stream particularly thoseproperties that cannot be reasonably rectified. As will be explainedsubsequently, the subject method is normally used in conjunction withfurther treatment since, under the conditions of complex formation, thelevel of anaphylatoxins normally increases.

The binding component will be nondiffusively bound, usually covalentlybound, to a membrane surface, where the membrane will be composed of aplastic, e.g. cellulosic or polystyrene, biocompatible material. Whileother structures may find application, including structures such asporous beads, hollow fibers or the like, membranes may be used toadvantage. In selecting a material, the selection will be based onbiochemical compatibility, ease of functionalization, level offunctionalization, degree of non-specific binding, with or without priortreatment, ease of fabrication, and the like.

Materials that may find application include nitrocellulose, cellulose,cellulose ester, e.g. acetate, nylon, polypropylene, polyethylene,silicone, polycarbonate, polyester, polyterephthalate, etc., orcombinations thereof. The membranes will usually have pores in the rangeof about 1 to 500 μ, more usually in the range of 2.5 to 25 μ. Aplurality of membrane layers will be employed. The layers may be ingroups stacked one upon the other, where the stacks will have at leasttwo membrane layers and may have ten or more membrane layers. Themembrane stacks will be separated so as to allow for the relatively freeflow of fluid through the membranes, while providing for a high surfacearea to ensure contact of the blood components with the bound component.Alternatively, a continuous spiral roll of membrane may be employed,where the flow is normal to a plane cutting through the spiral. Or, afluted membrane may be used, where the fluted layers may be packedtogether about a central core or in parallel structures.

Depending upon the volumes to be treated, the surface area of poroussurface will generally be in the range of about 0.2 to 3 m², moreusually in the range of about 0.3 to 2.5 m². With a substantially cellfree fluid, the rate of flow can be varied without concern as to celllysis. Flow rates will generally be in the range of about 0.001 to 0.2L/min, more usually in the range of about 0.002 to 0.1 L/min. Usually,the volume to be treated will be at least about 50 ml, more usually atleast about 250 ml, and preferably at least about 500 ml.

The weight ratio of bound specific binding member to membrane supportwill vary widely depending upon the nature of the specific bindingmember. For the most part, protein binding members will be in the rangeof about 0.5 to 50, more usually about 1 to 20 mg/gram of membrane. Theweight of binding member to volume of treated fluid may also be variedwidely depending upon the specific binding member, but will generally bein the range of about 0.05 to 10, more usually about 0.1 to 5 mg/ml. Ofcourse, depending upon the amount of the component which may be presentin the fluid stream, larger or smaller amounts may be necessary toensure that saturation is not achieved and that the capacity of thebinding component bound to the membrane is sufficient for the amount ofthe fluid component which will be encountered.

One can provide for a tortuous path by having membrane pack or stackseparators, which are effectively U-shaped and alternate in direction.Thus, the fluid flow would then be alternately redirected, so that thefluid will flow throughout the membrane pack in one direction, whileflowing through the successive membrane pack in the opposite direction.The distance the fluid flows may vary widely depending upon the purposeof the treatment but will usually be a distance of at least about 10 cmto 220 cm, more usually about 20 cm to 40 cm.

The binding component will be bound to the support in a manner whichminimizes leaching from the support during use and recovery of fluidcomponents bound to the specific binding member. The manner of bindingwill, therefore, normally be covalent, where the surface of the membraneis functionalized. Various techniques for functionalization existinvolving functionalized surfaces which may react with amines,carboxylic acids, activated aromatic rings, such as phenols as intyrosines, active heterocycles, such as histidine, or the like. Withsaccharides, either as the membrane or the binding component, thesaccharide may be cleaved to provide a dialdehyde which may then becondensed with an amine under reductive amination conditions. Theresulting aliphatic amine linkage provides for a strong, non-cleavablelinkage, which allows for repeated reuse of the membrane, whereby bloodcomponents may be isolated and released from the membrane efficientlyand in good yield. Where a polystyrene surface is employed, the surfacemay be functionalized using Freidel-Craft conditions forhalomethylation, nitration, with subsequent reduction to amino groups,or the like, where the Freidel-Crafts reaction is carried out intetramethylene sulfone or dimethylsulfoxide, particularly in thepresence of under about 1% by volume of water. See, for example,copending application Ser. No. 051,917 filed May 19, 1987. Thedisclosure of this application is specifically incorporated herein byreference. Aromatic amino groups may be diazotized and used to form adiazo bridge to tyrosines or triazines. The triazines are a relativelyunstable link and will usually not be employed.

Beside the membrane packs, other designs may be employed which providefor a large surface area for contacting the fluid stream. As alreadyindicated, one could employ a membrane as concentric tubes or spirallywound around a core, where the flow would be parallel to the surface ofthe cylinder. One could provide for flow throughout the membrane in asingle direction or have the flow be diverted in the opposite directionone or more times to greatly extend the path.

Instead of a membrane wound around a core, one could provide for afluted membrane, where the folded membrane provides for exposure of themembrane surface to the fluid stream. Any technique to provide forcomplete exposure of the binding component on the surface, so that allthe fluid is exposed to the opportunity for binding, while at the sametime providing for efficient use of space and a low probability ofclogging, may be employed. However, the use of the membrane sheet devicehas been found to be successful in providing a safe and efficient meansfor treating plasma and is, therefore, preferred.

It is found that when using the subject device, the formation ofspecific binding protein complexes results in a great enhancement incomplement activation to anaphylatoxins. Since the anaphylatoxins can bedetrimental if the plasma is restored to the host, the subject devicewill normally be employed with means for reducing a dangerous level ofanaphylatoxins to a safe level. It is found that this can be achieved bypassing the fluid from the subject device to a chamber containingsilicic acid particles, where the silicic acid is found to substantiallyremove dangerous levels of anaphylatoxin, without a significantlydeleterious effect on other components of the blood or the plasmacharacteristics. The anaphylatoxin removing device is described incopending U.S. application Ser. No. 191,039, filed May 6, 1988. Thedisclosure of this application is specifically incorporated herein byreference.

Briefly, the silicic acid particles will generally be of a size in therange of about 50 to 500 μ, and of neutral and acidic pH in the range ofabout 3 to 7. The pore size will generally be in the range of about 50to 350 Å with a surface area of at least about 200 m² /g. The fluidstream, particularly plasma, from the subject device will generally bedirected directly into the anaphylatoxin removing device.

The ratio of silicic acid to fluid generally will be in the range ofabout 10 to 100 g/L of fluid, more usually from about 15 to 50 g/L offluid. The temperature will generally range from about 20° to 40° C.,preferably from about 25° to 37° C. Ambient temperatures will usually beconvenient.

To illustrate the subject invention, a cellulose acetate membrane may beemployed. The membrane is a coating of cellulose acetate on an inertpolyester support. The coating may vary in thickness, generally being atleast about 100 μm to 200 μm, where the total thickness will range fromabout 150 to 500 μm, preferably about 150 to 300 μm. The celluloseacetate coating may be coated onto the support by any convenient means,using an appropriate volatile physiologically acceptable solvent. Thecellulose acetate coating may then be activated in accordance withconventional techniques, for example, dilute periodate to provide forthe desired level of activation. See, for example, U.S. Pat. Nos.4,299,916 and 4,391,904.

The membrane may then be flushed with a dilute solution of a protein tobe conjugated, e.g. protein A or acetylcholine receptor, where thesolution may be perfused through the membrane pack for sufficient timeto insure that the reaction has gone to completion and substantially allof the aldehyde groups have reacted. After mild flushing or perfusingwith an appropriate buffer, conveniently a buffer more dilute than thebuffer employed with the protein, so that non-covalently bound proteinis removed, the membrane may then be reacted with a dilute borohydridesolution to reduce the Schiff's bases or imines which were formed, so asto provide methyleneamines. Usually, a concentration of about 0.1 to 1Mborohydride may be employed in appropriate dilute buffer, e.g. boratebuffer. To insure the complete reaction, the reduction may be repeatedone or more times, each time washing with dilute buffer after thereductive treatment.

Finally, the device may be treated with dilute saline of at least above0.5M and not more than about 2M, followed by treatment with glycine ofabout 0.1 to 1M at an elevated temperature in the range of about 30° to50° C. resulting in the complete removal of any residual borohydride andany protein which has not become covalently bonded. After flushing withPBS, the membrane is stabilized with dilute glycerol, generally at about0.1 to 1% and may then be dried by any convenient means, e.g.centrifugation.

Any composition containing a lysine may be linked to the membrane in themanner described above. Thus, the above procedure provides for a simpleand efficient technique for binding lysine containing compounds to acellulosic surface in a manner which results in a low level of leaching,so that the bound protein does not contaminate any product which isextracted from the plasma and then eluted from the surface.

Instead of a cellulosic membrane, polystyrene or other biocompatiblearomatic containing plastic may be used in the form of small particleswhich may be functionalized at the surface employing a nitrating mediumin a tetramethylenesulfone solution in the presence of a small amount ofwater as described in copending application Ser. No. 051,917. Theresulting nitrated polystyrene may then be reduced to amino groups, soas to have a plurality of aniline groups on the surface of theparticles.

Antibodies, for example, may be oxidized in accordance with knowntechniques with periodate to provide for the dialdehydes as describedpreviously for the cellulose acetate. Following the procedure describedabove for reductive amination between lysines of a protein and theactivated cellulose surface, the activated antibodies may be combinedwith the particles to provide for reductive amination and antibodybinding to the particles. In this manner, particles having antibodiesbound to the surface in high concentration and functionalized so as tohave the binding sites available can be produced.

After the plasma has circulated through the device and, as appropriate,been further treated to remove any anaphylatoxins, it may then berestored to the host in accordance with conventional techniques. Thus,the plasma may be removed and returned in a continuous manner ordiscontinuous manner, as appropriate.

The device containing the bound components may then be used in a varietyof ways. The device may be restored by eluting the extracted componentusing an appropriate solution, such as a urea solution of about 2 to10M, dilute acetic acid of about 0.1 to 0.8M, guanidinium salts of about1 to 3M, or into 1 to 5M MgCl₂ or the like. The particular choice ofeluent will vary depending upon the material of interest, whether thematerial is to be recovered or discarded, the manner in which it may besubsequently used, or the like.

The subject device allows for collection of immune complexes, where theantigen may be identified and used in a variety of ways. The antigen maybe used to identify a particular disease, to produce antibodies, used inother devices for monitoring the presence of antibodies, or forgenerating antigen-specific immune effector or regulatory cells. Theantibodies may also be used for producing anti-idiotypes, to identifyantigens in other samples, for sequencing, so as to produce probes toidentify genes or mRNA in cells, or the like.

To further understand the subject invention, the drawings will now beconsidered. In FIG. 1, a schematic of the subject device is provided.The device 10 receives blood from one arm 12 of a patient throughconduit 14. Conduit 14 introduces the blood into the first chamber 16,where one or more components may be exchanged, removed, or otherwisemodified. The blood exits into conduit 20 and is directed by conduit 20to anaphylatoxin removal chamber 22. The modified blood free of anundesirable level of anaphylatoxins is then directed through conduit 24to the other arm 26 of the patient. In this manner, the blood has beenmodified in accordance with the needs of the patient and is returned tothe patient free of elevated levels of anaphylatoxins to avoid potentialshock.

In FIG. 2 is indicated an exploded view of a device in the shape of abox having first and second compartments where the first compartment hasa plurality of membranes overlying one another and the secondcompartment has the silicic acid. The membrane compartment provides foran alternating direction of flow of the blood derived stream through thecompartment. The device has a housing 30 with inlet 32 and outlet 34.Contained in the membrane compartment is O-ring 36, U-ring 40, andscreen 42. The U-ring controls the direction of flow of the stream. Ontop of the screen 42 is a second O-ring 44 which separates the O-ringfrom membrane pack 46. The membrane employed may be Nalgene affinitychromatography unit bound with protein A, U12A or U38A (cat. nos.751-2012 and 751-5038). The membrane pack will have a plurality ofmembranes lying one atop the other to which will be bound the specificbinding pair members. Conveniently, each pack may contain from about 5to 25, usually 5 to 20, membranes. The blood derived stream will pass upthrough the membrane pack 46 contacting the specific binding pairmembers and rising up through the pores to repetitively contact eachsucceeding membrane. Once the blood derived stream has passed throughthe membrane pack, the assemblage of O-ring 36, U-ring 40, positioned inthe opposite direction of the previous U-ring 40, O-ring 36, screen 42,second O-ring 44 and membrane pack 46 may be repeated one or more timesdepending upon the size of the unit, the amount of material to beextracted, the binding capacity of the membrane packs and the like. Theparticular component which is the last component is not critical to thisinvention.

Various biocompatible materials may be employed for the variouscomponents. Conveniently, the O-ring and U-spacers may be high densitypolypropylene, the screens polypropylene and the housing polycarbonate.

Surmounting the components of the membrane compartment will be an innerlid 50 having port 52. The port 52 will be of approximately the samedimensions as the inlet and outlet ports 32 and 34 respectively of thehousing 30. A polyethylene filter, not shown, conveniently of a poresize of 35-60 μ is applied across the port to prevent access of silicicacid particles into the membrane pack compartment. Barriers 54 and 60are employed to maintain the silicic acid within a predetermined area inthe silicic acid compartment. The silicic acid particles are indicatedas a box 62. The silicic acid particles may be of a size in the range offrom about 50 to 300 μ. A cover 64 is then used to close the housing 30completing the device.

A third device is depicted in FIGS. 3 and 4. The device 70 iscylindrical, having cylindrical membrane 72 fitted into cylinder 74which serves as the membrane compartment. An inner tube or sleeve 76serves for mounting the membrane 72 and to define the silicic acidcompartment 80. Silicic acid particles 81 as described previously arethen packed into silicic acid compartment 80. First and second screens82 and 84 respectively are mounted at the bottom and top of compartment80 to ensure that silicic acid particles do not escape.

The device may be assembled by employing top cap 85 and bottom cap 90and mounting inner tube 76 on projection 86 which holds the inner tube76 in place. Included within inner tube 76 is mounting 90 which includesconduit 92, which is in alignment with orifice 94 in innertube 76.Mounting 90 receives and holds first and second screens 82 and 84 inposition to prevent the silicic acid particles 81 from entering themembrane compartment 74. The top cap 85 has plasma inlet 96 and plasmaoutlet 100.

After mounting the inner tube 76 on projection 86, membrane 72 is thenfitted onto inner tube 76, followed by mounting of membrane compartmenttube 74 which encloses membrane 72. Assemblage of the device iscompleted by adding the upper silicic acid screens 82 and 84 over thesilicic acid, followed by enclosing the device with top cap 85 whichincludes plasma inlet orifice 96 and plasma outlet 100.

The top of the membrane may be coated with netting 102 which is held inplace with a hot melt 104 so as to provide structural stability to themembrane 72.

In using the device, the plasma will enter inlet 96 and flow downwardlythrough membrane 72. The flow of plasma will be circular around thedevice, filling the membrane with the plasma. The plasma will reach thebottom of the device and pass through orifice 94 into conduit 92. Fromconduit 92, the plasma will pass through first and second screens 82 and84 into the silicic acid particles 81, where anaphylatoxins will beremoved. After passing upwardly through the silicic acid particles 81,the plasma will pass through upper screens 82 and 84 through outlet 100.

The membrane may then be easily removed for regeneration or other use byremoving the top cap 85 and the membrane compartment 74 and retrievingthe membrane 72 by removal from the sleeve or inner tube 76.

Other equipment may be employed with the device, including additionalextraction systems. Usually, a pump or an hydraulic device will beemployed to move the blood from the patient or other source through thevarious compartments and conduits. Various alarm and control systems maybe employed for detecting rate of flow, flow blockages, air bubbles,clots, or the like. Other components may be additional filters,absorbents, chemical treatments, radiation treatments, and the like.Various electronic equipment may be associated with the device toprovide for the automation of various fluid flows, the eluent where thebound material is removed, and the like.

The following examples are for illustration and not by way oflimitation.

The device as depicted in FIG. 1 is prepared as follows. A polycarbonatecase encloses ten packs of cellulosic membranes, each pack contains tenrectangular sheets of membrane. Each pack is separated from the next bya polypropylene O-ring and a flow-directing U-ring, as well as thepolypropylene separation screens. The ten packs are compressed beneath apolycarbonate lid having an access port equal in diameter to the inletand outlet ports of the polycarbonate case. A 35-60 μ high-densitypolyethylene filter is applied across the access port to prevent entryof silicic acid particles into the compartment containing the membranepacks. The same filter is placed across the outlet of the silicic acidcompartment to prevent the silicic acid from being entrained with theplasma.

The silicic acid has a particle size of 75-250 μ. The polycarbonate lidis then applied.

The receptor is then covalently immobilized to the membrane as describedbelow. By the process of immobilization described below, approximately400 mg of recombinant protein A (rPA) is covalently attached to themembrane prior to the addition of the silicic acid to the silicic acidcompartment. In this manner the subject device contains ten square feetof cellulosic membrane to which is bound approximately 400 mg rPA.

The membrane is prepared by solution casting of cellulose acetate onto apolyester support matrix. The membrane (B10-38) is manufactured byMemtek Corp., Bellenia, Mass. The membrane employes a cellulose materialreinforced with a polyester (Dacron) spun-bonded material. Thereinforcing web is incorporated in the membrane during membraneformation and is an intergral part of the membrane but does notsignificantly affect the membrane properties. The membrane has a poresize distribution predominantly in the 1 to 1.5 um range, as representedby the foam point. The membrane is dried, wound on a plastic core, andquality control tested. The membrane is then processed through a seriesof chemical washes and surface activated by periodate oxidation. Themembrane is then thoroughly washed and stabilized with a solution ofglycerin and sterile water. After drying, the membrane may be packagedfor subsequent use. The ratio of bound rPA to weight of membrane isabout 5.5 mg rPA/gram of membrane.

The membrane is further characterized by having a width of about3.18-3.25 inches, a length of 6.42 to 6.52 inches, a thickness of175-250 μm, a weight of about 90-125 mg/47 mm disc, a water flux(ml/min/cm²) of about 20-25 inches of Hg, vacuum, and a human IgGbinding capacity of about 70 g/cm². The membranes are substantiallynon-pyrogenic and non-toxic.

Immobilization of protein is achieved as follows. After the dry weightis recorded and the device purged with carbon dioxide, the device isflushed with 0.2M borate buffer. A 12.6 mg/ml rPA solution in 0.5Mborate buffer, pH 9.2, is circulated throughout the device for 11-15hours at room temperature, followed with flushing with 0.2M boratebuffer. The device is then profused with 1.0 mg/ml sodium borohydride in0.5M borate buffer flushing with 0.2M borate buffer, followed byrepeating the borohydride and flushing steps. After flushing the devicewith 1M sodium chloride at 35° C., the device is flushed with 0.5Mglycine-HC1 at 35° C. Glycine-HC1 (0.5M) is then circulated at 37° C.until the effluent has an optical density of less than about 0.001 at280 nm. The device is then flushed successively with phosphate bufferedsaline to a pH of 7.0-7.2, while monitoring to ensure that the opticaldensity remains at the previously defined level, followed by flushingwith 0.5% glycerol. The device is then centrifuged at 2,000 rpm for 30min to facilitate drying, incubated at approximately 40° C. whileperfusing with filtered air or nitrogen until the dried weight of eachunit is within approximately 10 g of the initial dry weight.

In order to determine whether the covalently bound protein issubstantially free of leaching when perfused with plasma or anotheraqueous medium, the following experiments were carried out. Fourethylene oxide-sterilized prototype devices having polyethylene silicicacid filters, ten square feet of membrane, 400 mg of rPA and 90 g ofsilicic acid were subjected to the following flush/perfusion protocol.Two 500 ml volumes of 0.9% saline at room temperature were pumpedthrough the device at 200 ml/min and collected separately. A sample wastaken of each. Subsequently, two additional 500 ml volumes of 0.9%saline at 37° C. were sequentially pumped through each device at 200ml/min and collected separately. A sample was taken of each.

An additional 500 ml of 0.9% saline at 37° C. was recirculatedcontinuously through the device at 50 ml/min for 4 h. At the end of thisprocedure, the saline perfusate was collected separately and a sampletaken for assay. All samples were assayed by an rPA specific ELISA assayand by less specific methods (BCA, biocinchoninic acid, and opticaldensity at 210 and 280 nm). The results are indicated in Table 1.

The BCA active material was established to be a contaminant of theassembly used in the flush/perfusion protocol by employing a shamprocedure using a tubing and pump assembly identical to theflush/perfusion study, except that the device was not included in theloop. The data showed that negligible (<0.001%) amounts of rPA weredetected in eluates from the device during perfusion.

To determine the specificity of binding of the subject device, thefollowing experiments were carried out. Two devices were studied, onewith silicic acid and one without silicic acid, where a polyethylenefilter was used as described previously. All the devices were sterilizedwith ethylene oxide. The protocol was as follows.

After priming the device with 1L of a phosphate buffer saline flush,1.5L normal human plasma was pumped through the device at 50 ml/min in asingle pass. Pre- and post-perfusion plasma samples were retained.Earlier data had shown that protein A is saturated by approximately1-1.5L of normal plasma. Two liter phosphate buffered saline flusheswere then followed by two 0.5M acetic acid 2L flushes and samples of theacetic acid washes saved. Both the acetic acid wash samples and thepre/post-plasma were

                  TABLE 1                                                         ______________________________________                                        LEACHING OF PROTEIN A BY FLUSH/PERFUSION                                      Four devices having internal polyethylene                                     silicic acid filters.                                                         Device                                                                        Item            ELISA    BCA   210 nm  280 nm                                 Number Sample   (ng/ml)  (mg)  (OD units)                                                                            (OD units)                             ______________________________________                                        1987   Flush 1  <10      3.4   .274    .031                                          Flush 2  <80      0     .131    .014                                          Flush 3  85       0     .090    .012                                          Flush 4  10       0     .056    .009                                          Perfusion                                                                              96       1.55  .242    .023                                   1990   Flush 1  10       0     .442    .036                                          Flush 2  <80      0     .141    .020                                          Flush 3  83       0     .084    .017                                          Flush 4  11       0     .058    .013                                          Perfusion                                                                              109      0     .144    .023                                   2000   Flush 1  12       0     .178    .089                                          Flush 2  <80      0     .098    .016                                          Flush 3  <80      0     .060    .009                                          Flush 4  12       0     .034    .006                                          Perfusion                                                                              80       0     .116     .01                                   2004   Flush 1  11       0     .186    .014                                          Flush 2  <80      0     .116    .004                                          Flush 3  115      0     .055    .005                                          Flush 4  13       0     .039    .001                                          Perfusion                                                                              80       1.35  .107    .007                                   ______________________________________                                    

analyzed by a Bradford assay kit (Bio-Rad 500-001). The differencebetween pre- and post- samples minus that contained in residual plasmarepresents total IgG binding, while the specifically bound IgG is theresult obtained from the acetic acid washes. This is also therecoverable IgG.

Table 2 indicates the results.

                  TABLE 2                                                         ______________________________________                                        IgG BINDING BY PROTOTYPE DEVICES                                              Device               Specificity                                                                             Total IgG                                      Item                 Binding   Binding                                        Number               (grams)   (grams)                                        ______________________________________                                        A. IgG binding by 15 ft.sup.2  device having 750μ rPA* but                 no silicic acid compartments                                                  1767                 0.874     2.64                                           1768                 0.910     3.51                                           1747                 1.015     1.49                                           1753                 0.80      7.50                                           1734                 1.246     5.05                                           1737                 1.326     3.96                                           1718                 1.540     3.00                                                     mean       1.10      3.88                                           B. IgG binding by assembled device having polyethylene                        filters (10 ft.sup.2, 400 mg rPA, 90 gm silicic acid).                        1987                 1.037     2.685                                          1990                 0.959     2.550                                          2000                 0.805     3.735                                          2004                 0.912     2.390                                                    mean       0.928     2.84                                           ______________________________________                                         *rPA = recombinant Protein A.                                            

The above results demonstrate that the subject device, with or withoutthe silicic acid compartment, effectively and consistently binds IgGfrom human plasma. Based on the above data, a mean value ofapproximately 3.0 g total IgG is bound from 1500 ml of processed plasma.

In another study, the preference for immune complex over uncomplexedimmunoglobulin was established by monitoring the complex and monomerbefore and after passage through the device. As the results show, thebound rPA finally became saturated with immune-complex which displacedthe initially bound monomer.

To further evaluate the effectiveness of the device, the acetic acidwashes were concentrated, the concentrated eluate desalted and appliedto an IEP plate and developed with antibodies against IgG, IgM and wholeserum proteins. The results showed that only IgG and IgM were present inthe eluate with no other plasma proteins present. Thus, only thoseligands which specifically bind to the rPA receptor were bound andremoved from the plasma through the device.

Another area of concern is platelets. For the most part, platelets willnot be efficiently removed by the usual technique employed for removalof cells from blood, namely centrifugation. Therefore, the device wastested to determine whether platelets would clog the device and whateffect the device would have on the viability and state of theplatelets.

During four routine plasma exchange procedures on the IBM Model 2997Blood Cell Separator, the device was connected on-line between theplasma pump and filtrate collection bag. Three way stop cocks werespliced in line just before and just after the device to permitpre-device and post-device sampling. Plasma samples, as pairedpre/post-samples, were collected at 5, 25 and 45 min into the exchangeprocedure. These times correspond with approximate exchange volumes of0.5, 1.0 and 1.5L from a total of 3L procedures. To determine theplatelet-device interaction, the following measurements were made onpaired samples: platelet count; beta-thromboglobulin (BTG) level; andintraplatelet serotonin level. After each procedure, the device weregrossly examined for evidence of platelet "clumping" and aggregation.

Tables 3 through 6 indicate the results using a 15 ft² device withapproximately 500 mg rPA bound and without a silicic acid compartment.

These results show that in a fully functional device (I_(g) G was boundby the membrane) although some platelets are bound in the device, thefluid path is not affected (plasma continues to flow freely), theplatelets bound in the device are not activated (minimal changes inbeta.thromboglobulin levels) and the platelets passing through thedevice are fully functional (no change in intra-platelet serotoninlevels). The device is thus neutral with respect to platelet function.

                  TABLE 3                                                         ______________________________________                                        PLATELET COUNTS IN PLASMA PRE/POST DEVICE                                     AT VARIOUS TIME INTERVALS                                                     Device Patient  Time                                                          Item   Baseline Into      Pre    Post   %                                     Number Count    Exchange  Device Device Change                                ______________________________________                                        1508   302,000   5 min.   61,000 30,000 -51%                                                  25 min.   85,000 51,000 -40%                                                  45 min.   59,000 40,000 -32%                                  1509   418,000   5 min.   96,000 30,000 -69%                                                  25 min.   59,000 35,000 -41%                                                  45 min.   30,000 29,000  -3%                                  1510   458,000   5 min.   695,000                                                                              457,000                                                                              -34%                                                  25 min.   94,000 61,000 -35%                                                  45 min.   344,000                                                                              295,000                                                                              -14%                                  1511   217,000   5 min.   52,000 76,000 +46%                                                  25 min.   46,000 27,000 -41%                                                  45 min.   37,000 20,000 -46%                                  ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        BETA THROMBOGLOBULIN* MEASUREMENT IN                                          PLASMA ENTERING AND EXITING THE DEVICE                                        Device    Time          BTG (ng/ml)                                           Item      Into          Pre-    Post-                                         Number    Exchange      Device  Device                                        ______________________________________                                        1508       5 min.       36      ND**                                                    25 min.       32      123                                                     45 min.       32      170                                           1509       5 min.       ND      ND                                                      25 min.       41       45                                                     45 min.       46       64                                           1510       5 min.       ND      ND                                                      25 min.       96      100                                                     45 min.       88      210                                           1511       5 min.       18       26                                                     25 min.       28       64                                                     45 min.       32      108                                           ______________________________________                                         *Normal plasma range for BTG is 24-28 ng/ml                                   **ND = not determined. Certain samples were omitted from test as only         enough reagent for 19 evaluations was available.                         

                  TABLE 5                                                         ______________________________________                                        INTRAPLATELET SEROTONIN LEVELS IN PLATELETS                                   FROM PLASMA PRE- AND POST-DEVICE                                                                    Intraplatelet Seratonin                                 Device   Time         Levels (ng/10.sup.9  platelets)*                        Item     Into         Pre-     Post-                                          Number   Exchange     Device   Device                                         ______________________________________                                        1508      5 min.      250      250                                                     25 min.      235      235                                                     45 min.      230      225                                            1509     5 min.       140      140                                                     25 min.      145      140                                                     45 min.      290      290                                            1510      5 min.      110       90                                                     25 min.       80       80                                                     45 min.       55       55                                            1511      5 min.      211       86                                                     25 min.      270      270                                                     45 min.      230      230                                            ______________________________________                                         *Normal range is 300-980 ng/10.sup.9  platelets                          

                  TABLE 6                                                         ______________________________________                                        TOTAL PROTEIN FROM PLASMA                                                     ENTERING AND EXITING THE DEVICE                                               Device    Time                                                                Item      Into          Pre-    Post-                                         Number    Exchange      Device  Device                                        ______________________________________                                                          Total Protein                                                                 (gm/100 ml)                                                 1508       5 min.       4.8     2.5                                                     25 min.       4.2     4.0                                                     45 min.       4.2     3.9                                           1509       5 min.       5.5     4.2                                                     25 min.       5.8     5.5                                                     45 min.       5.8     5.8                                           1510       5 min.       4.3     3.0                                                     25 min.       4.8     4.6                                                     45 min.       5.2     4.8                                           1511       5 min.       5.2     3.2                                                     25 min.       4.5     4.2                                                     45 min.       4.0     4.0                                                             IgG Measurement                                                               (gm/L)**                                                    1508      25 min.       5.55    4.18                                          ______________________________________                                         *Drop in this sample mainly due to dilution.                                  **Normal range is 8-18 gm/L.                                             

The next study was to demonstrate the device could be used to removespecific proteins from plasma for example tumor necrosis factor (TNF)which is involved in septic shock.

Outdated normal human plasma was employed. The plasma was divided into2×1L volumes and 17 μg of ³⁵ S-TNF was added to each liter to give atotal 5.1×10⁶ cpm/L. One ml of each preparation was counted in a 5 mlOptofluor to obtain a pre-perfusion radioactivity measurement. The TNFhad been labeled with ³⁵ S-methionine in accordance with conventionaltechniques. Counting of input and output plasma samples by liquidscintillation provided direct measurements of TNF in the plasma Thedevice had a 4 ft² path with monoclonal α-TNF bound as described forrPA. There was no silicic acid compartment. The amount of TNF bound bythe device was derived by subtracting the amount remaining in the plasmaafter perfusion (output plasma) from the amount of TNF in the inputplasma. The fraction of immunoactive radio-labeled TNF was determinedusing a solid-phase radioimmunoassay in which excess antibody wasimmobilized. Briefly, 10, 25, 50, 100, 125, 150 and 200 μl of a 100μg/ml stock of an anti-TNF monoclonal antibody in 0.5M carbonate buffer(pH 9.0) were added to Removawells (Dynatech Immulon I) and incubatedovernight at 4° C. The wells were blocked with 200 μl 5% BSA in thecarbonate buffer for 2 h at room temperature. The wells were washed aminimum of 6× with PBS and 5 or 50 μg of ³⁵ S-TNF in 200 μl of PBS wasadded to each well and the wells incubated at room temperature for 1.5h. At the end of the incubation, the solution in each well wastransferred as completely as possible to a scintillation vial andcounted. The individual wells were also counted. The percent CPM bound(CPMs on wells/(CPMs on wells 30 CPMs in solution) ×100% was plottedagainst the amount of adsorbed anti-TNF monoclonal antibodies/well. Thepercent immunoactive ³⁵ S-TNF was then taken as the plateau value fromthe above plot. These results provided for the binding activity of the³⁵ S-TNF.

The plasma containing ³⁵ S-TNF was passed through the preiouslydescribed device (a device having bound human IgG was employed as acontrol) at a flow rate of 50 ml/min for 3 h at room temperature. At theend of 1, 2, and 3 h of perfusion, 1 ml aliquots were withdrawn from theplasma reservoir and counted. Each device was perfused with 5×500 mlsaline for 10 min at room temperature at a flow rate of 150 ml/min. Oneml aliquots of each rinse were counted. The devices were then elutedwith up to 5×100 ml 4M magnesium chloride. After each elution, 1 mlaliquots were withdrawn and counted. Tables 7 and 8 indicate theresults.

                  TABLE 7                                                         ______________________________________                                        DEPLETION OF TNF FROM PLASMA BY DEVICE                                        Anti-TNF Device     Control Device                                                             Immuno-            Immuno-                                                    reactive           reactive                                         TNF       .sup.35 S-TNF                                                                            TNF     .sup.35 S-TNF                                    Removed.sup.1                                                                           Removed.sup.1, 2                                                                         Removed.sup.1                                                                         Removed.sup.1, 2                          Sample (μg)   (%)        (μg) (%)                                       ______________________________________                                        1 hr   7.5       68.8       1.1     10.1                                      2 hr   7.8       71.6       0.9     8.3                                       3 hr   8.1       74.3       0.3     2.8                                       ______________________________________                                         .sup.1 Cumulative values                                                      ##STR1##                                                                 

                  TABLE 8                                                         ______________________________________                                        RECOVERY OF BOUND TNF                                                         FROM DEVICE BY 4 M MgCl.sub.2 ELUTION                                                  Anti-TNF Device  Control Device                                      4M MgCl.sub.2                                                                            TNF    Recovery.sup.1                                                                            TNF  Recovery.sup.1                             Elution    (μg)                                                                              (%)         (μg)                                                                            (%)                                        ______________________________________                                        1st Eluate 2.0    24.7        0.2  40.0                                       2nd Eluate 2.3    28.4        0.1  20.0                                       3rd Eluate 1.5    18.5        0.0   0.0                                       4th Eluate 0.6    7.4         --   --                                         5th Eluate 0.2    2.5         --   --                                         Total      6.6    81.5        0.3  60.0                                       ______________________________________                                         .sup.1 Values calculated from                                                 ##STR2##                                                                 

Based on binding activity, it was found that the immunobinding activityof ³⁵ S-TNF was about 64.1%. This immunoreactivity level was thenfactored into the subsequent calculations from results of plasmaperfusion and magnesium chloride elution. The above tables show that74.3% TNF was removed by the anti-TNF device from 1L plasma containing 1nm TNF in a 3 h perfusion. By comparison, the control device removedonly 2.8% TNF. In addition, the results show that 81.5% of bound TNF wasrecovered from the device.

In the next study, the removal of anti-DNA antibody from the sera ofpatients with systemic Lupus Erythematosus is investigated. Theadsorbent employs polystyrene beads (500 μ) which are prepared asdescribed in application Ser. No. 051,917, filed May 19, 1987. Calfthymus DNA (10 mg/ml) is sonicated using a fine-tipped probe over 1 h,using a pulsed delivery. The sheared DNA has an average size of about500-1500 bp. The sheared DNA is then extracted 2× with phenol-chloroformand precipitated with ethanol. The sheared DNA is then resuspended to 10mg/ml and stored in 5 ml aliquots at -20° C. until used. Polystyrenebeads (Precision Plastic Ball beads) in cold 0.05M sulfuric acid (5 ml)are added to a cold 50 ml polypropylene centrifuge tube, the supernatantremoved and 45 ml of cold 2% sodium nitrite in 1M HC1 added. Aftermixing on a platform rocker for 30 min at 4° C., the beads are collectedon a cold 15 ml sintered glass filter using vacuum suction, washed with100 ml cold 0.5M H₂ SO₄, followed by 200 ml cold water. The drainedbeads are then added to a cold 50 ml polypropylene centrifuge tube,followed by the addition of 10 ml of 5 mg/ml ³² P-DNA in cold 0.025Mborate buffer, pH 9.2 and the mixture incubated overnight at 4° C. on aplatform rocker. Supernatant is transferred to a 10 ml polypropylenetube, the labeled beads washed 15× with 50 ml water, 2× with 2M sodiumchloride and 15× with water. A 0.125 ml aliquot of beads is counted toprovide 40,000 cpm in 5 ml packed volume of the DNA beads.

By replacing the membranes with the subject beads and perfusing thebeads in the manner described previously, with plasma samples frompatients having anti-DNA, the anti-DNA antibody will bind to the DNA, toprovide a plasma effluent substantially free of autoantibody.

It is evident from the above results, that the subject devices have awide variety of applications, in diagnosis and therapy, and in providingfor a source of modified plasma. Despite the harsh conditions employedfor fluid flow, high efficiencies are obtained in the removal ofspecific components, while at the same time little if any loss isobserved of the materials bound to the supports. In addition, highrecoveries are achieved from materials which are bound, substantiallyfree of other components of the streams passed through the device, aswell as free of the binding components bound to the support in thedevice.

The subject device can be used for therapeutic purposes, in removingmaterials adverse to a host, such as immune complexes, tumor necrosisfactor, and the like. In addition, the device may be used for diagnosis,by eluting and identifying the particular materials which have becomebound. In the case of immune complexes, the complexes may be isolatedand the antigen assayed to determine the source which provoked theimmune response. Thus, the subject devices have a multiplicity ofutilities and structures and provide a safe and effective way to modifyplasma and obtain information from the plasma which may be used indiagnosis and therapy.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A device for modifying plasma by removing orremodeling at least one plasma component, which plasma component is amember of a specific binding pair, said device having a predeterminedvolume rating, said device comprising:a container of a biocompatiblematerial having an entry and an exit port and an extended flow pathbetween said ports; a high surface area biocompatible cellulosic poroussolid support comprising a member of said specific binding pairsubstantially uniformly and irreversibly bonded to said support in anamount sufficient to bind the reciprocal member of said specific bindingpair at the volume rating of said device, wherein said flow path directssaid plasma through said porous support; said porous support comprisingmembrane sheets in stacks of at least two sheets, there being at leasttwo stacks; separating said stacks are alternating U-rings in alternatedirections to alternately direct the flow path in opposite directions.2. A device according to claim 1, wherein said cellulosic support is acellulose acetate coating on a polyester membrane carrier.
 3. A deviceaccording to claim 2, wherein said specific binding member is a proteinbonded to said support by reductive amination.
 4. A device according toclaim 3, wherein said protein specifically binds to immunoglobulin.
 5. Adevice according to claim 4, wherein said protein is recombinant proteinA.
 6. A device according to claim 3, wherein said protein is a membranesurface receptor.
 7. A device according to claim 6, wherein saidmembrane surface receptor is acetylcholine receptor.
 8. A deviceaccording to claim 2, comprising in combination in fluid receivingrelationship with said exit port a silicic acid anaphylatoxin removingcompartment.